Poliovirus is a small, iscosahedral RNA-containing picornavirus best known as the etiologic agent of paralytic poliomyelitis. Infection begins when virus is ingested and replicates in the gut, leading to a viremia. In a small number of infected individuals, virus invades the central nervous system from the blood. Lytic viral replication with motor neurons in the brain and spinal cord results in destruction of these cells and the characteristic flaccid paralysis of poliomyelitis [Bodian D., Science 12:105-108 (1955)].
Although during its viremic stage many tissues are exposed to poliovirus, replication is limited to the oropharyngeal and intestinal mucosa, the Peyer's patches of the ileum, and motor neurons within the central nervous system. Several experimental results support the suggestion that the restricted tissue tropism of poliovirus is a result of limited expression of specific viral attachment sites, or receptors. In binding studies using tissue homogenate, the poliovirus receptor is detected only in tissues that are sites of poliovirus replication [Holland, J. J. Virology 15:312-326 (1961)]. Furthermore, lack of susceptibility to poliovirus infection, both in primate and non-primate cell types, can be circumvented by introducing viral RNA into the cells by transfection, indicating that resistance to infection is due to a block in binding, entry, or uncoating of poliovirions [Holland, J. J., McLaren, J. C., and Syverton, J. T., J. Exp. Med. 110:65-80 (1959)]. Finally, the results of gene transfer experiments indicated the mouse L cells transformed with human DNA express poliovirus receptors at the cell surface and become susceptible to infection [Mendelsohn, C., Johnson, B., Leonetti, K. A., Nobis, P., Wimmer, E. and Racaniello, V. R., Proc. Natl. Acad. Sci. USA 83:7845-7849 (1986)].
Work in other viral systems strongly implicates cellular receptors in tissue tropism and pathogenesis. For example, the human T cell glycoprotein CD4 is the receptor for HIV-I [Maddon, P. J., Dalgeleish, A. G., McDougal, J. S., Clapham, P. R., Weiss, R. A. and Axel, R. Cell 47:333-348 (1986)]. Expression of the CD4 on T helper cells is though to be responsible for the selective infection and destruction of these cells observed in individuals infect with HIV-I Human CD4-negative cells, which are resistant to infection by HIV-I, can be rendered susceptible to infection by transfection with CDNA clones encoding the CD4 receptor.
Biochemical studies indicated that the poliovirus receptor is an integral membrane protein [Krah, D. L. and Crowell, R. L., Virology 118:148-156 (1982)]. However, it has not been possible to purify the receptor protein from membrane preparations using assays that require binding of virus or antibody, probably due to the liability of the respective binding sites. Several monoclonal antibodies have been isolated which inhibit the binding of poliovirus to cultured cells [Minor, P. D., Pipkin, P. A., Hockley, D., Schild, G. C. and Almond, J. W. Virus Res. 1:203-212 (1984); Nobis, P., Zibirre, R., Meyer, G., Kuhne, J., Warnecke, G. and Kock, G. J. Gen. Virol. 6:2563-2569 (1985); Shepley, M. P. Sherry, B. and Weiner, H. L. Proc. Natl. Acad. Sci, USA 85:7743-7747 (1988)]. Monoclonal antibody D171 competes with the 3 poliovirus serotypes for a common high affinity binding site on permissive cells and does not bind to cells that are resistant to poliovirus infection [Nobis et al., (1985)]. HeLa cells contain approximately 100,000 D171 binding sites (P. Nobis, personal communication) and 3000 poliovirus binding sites [Lonberg-Holm, K. and Philipson, L. Receptors and Recognition. (Chapman and Hall, London) (1981)], suggesting that virus binding is multivalent. A second type of monoclonal antibody partially blocks infection with poliovirus type 2 and to a lesser extent with poliovirus type 1, but has little effect on type 3 binding [Shepley et al., (1988)]. This antibody recognizes a 100 kd protein in the membrane of poliovirus-susceptible cell lines and human spinal cord, and specifically stains neurons at the neuromuscular junction.
The subject invention discloses isolated genomic and CDNA clones encoding poliovirus receptors from HeLa cells. Transformation of resistant mouse cells with either of two CDNA clones leads to expression of the receptor on the cell surface and susceptibility to poliovirus infection. Northern hybridization analysis indicates that a 3.3 kb receptor transcript is present in many human tissues, including kidney, which does not contain poliovirus binding activity and which is not a site of poliovirus replication. Thus, at least in the kidney, expression of poliovirus receptor RNA is not sufficient to permit viral infection.
The poliovirus receptor CDNA clones encode putative polypeptides of 43,000 and 45,000 daltons that contain identical extracellular and transmembrane domains, but differ at the cytoplasmic tails. Protein homology comparisons revealed that the poliovirus receptor is a new member of the immunoglobulin superfamily [For a review of the immunoglobulin superfamily see: Williams, A. F., and Barclay A. N. Ann. Rev. Immunol, 6:381-405 (1988)]. The extracellular portion of the receptor may be folded into a structure composed of 3 domains stabilized by intrachain disulfide bonds.